A major obstacle to the development of vaccines against viruses and bacteria, particularly those with multiple serotypes or a high rate of mutation, against which elicitation of neutralizing antibodies and/or protective cell-mediated immune responses is desirable, is the diversity of the external proteins among different isolates or strains. Since cytotoxic T-lymphocytes (CTLs) in both mice and humans are capable of recognizing epitopes derived from conserved internal viral proteins [J. W. Yewdell et al., Proc. Natl. Acad. Sci. (USA) 82, 1785 (1985); A. R. M. Townsend, et al., Cell 44, 959 (1986); A. J. McMichael et al., J. Gen. Virol. 67, 719 (1986); J. Bastin et al., J. Exp. Med. 165, 1508 (1987); A. R. M. Townsend and H. Bodmer, Annu. Rev. Immunol. 7, 601 (1989)], and are thought to be important in the immune response against viruses [Y.-L. Lin and B.A. Askonas, J. Exp. Med. 154, 225 (1981); I. Gardner et al., Eur. J. Immunol. 4, 68 (1974); K. L. Yap and G. L. Ada, Nature 273, 238 (1978); A. J. McMichael et al., New Engl. J. Med. 309, 13 (1983); P. M. Taylor and B. A. Askonas, Immunol. 58, 417 (1986)], efforts have been directed towards the development of CTL vaccines capable of providing heterologous protection against different viral strains.
It is known that CTLs kill virally- or bacterially-infected cells when their T cell receptors recognize foreign peptides associated with MHC class I and/or class II molecules. These peptides can be derived from endogenously synthesized foreign proteins, regardless of the protein""s location or function within the pathogen. By recognition of epitopes from conserved proteins, CTLs may provide heterologous protection. In the case of intracellular bacteria, proteins secreted by or released from the bacteria are processed and presented by MHC class I and II molecules, thereby generating T-cell responses that may play a role in reducing or eliminating infection.
Most efforts to generate CTL responses have either used replicating vectors to produce the protein antigen within the cell [J. R. Bennink et al., ibid. 311, 578 (1984); J. R. Bennink and J. W. Yewdell, Top. Microbiol. Immunol. 163, 153 (1990); C. K. Stover et al., Nature 351, 456 (1991); A. Aldovini and R. A. Young, Nature 351, 479 (1991); R. Schafer et al., J. Immunol. 149, 53 (1992); C. S. Hahn et al., Proc. Natl. Acad. Sci. (USA) 89, 2679 (1992)], or they have focused upon the introduction of peptides into the cytosol [F. R. Carbone and M. J. Bevan, J. Exp. Med. 169, 603 (1989); K. Deres et al., Nature 342, 561 (1989); H. Takahashi et al., ibid. 344, 873 (1990); D. S. Collins et al., J. Immunol. 148, 3336 (1992); M. J. Newman et al., ibid. 148, 2357 (1992)]. Both of these approaches have limitations that may reduce their utility as vaccines. Retroviral vectors have restrictions on the size and structure of polypeptides that can be expressed as fusion proteins while maintaining the ability of the recombinant virus to replicate [A. D. Miller, Top. Microbiol. Immunol. 158, 1 (1992)], and the effectiveness of vectors such as vaccinia for subsequent immunizations may be compromised by immune responses against vaccinia [E. L. Cooney et al., Lancet 337, 567 (1991)]. Also, viral vectors and modified pathogens have inherent risks that may hinder their use in humans [R. R. Redfield et al., New Engl. J. Med. 316, 673 (1987); L. Mascola et al., Arch. Intern. Med. 149, 1569 (1989)]. Furthermore, the selection of peptide epitopes to be presented is dependent upon the structure of an individual""s MHC antigens and, therefore, peptide vaccines may have limited effectiveness due to the diversity of MHC haplotypes in outbred populations.
Benvenisty, N., and Reshef, L. [PNAS 83, 9551-9555, (1986)] showed that CaCl2 precipitated DNA introduced into mice intraperitoneally (i.p.), intravenously (i.v.) or intramuscularly (i.m.) could be expressed. The intramuscular (i.m.) injection of DNA expression vectors in mice has been demonstrated to result in the uptake of DNA by the muscle cells and expression of the protein encoded by the DNA [J. A. Wolff et al., Science 247, 1465 (1990); G. Ascadi et al., Nature 352, 815 (1991)]. The plasmids were shown to be maintained episomally and did not replicate. Subsequently, persistent expression has been observed after i.m. injection in skeletal muscle of rats, fish and primates, and cardiac muscle of rats [H. Lin et al., Circulation 82, 2217 (1990); R. N. Kitsis et al., Proc. Natl. Acad. Sci. (USA) 88, 4138 (1991); E. Hansen et al., FEBS Lett. 290, 73 (1991); S. Jiao et al., Hum. Gene Therapy 3, 21 (1992); J. A. Wolff et al., Human Mol. Genet. 1, 363 (1992)]. The technique of using nucleic acids as therapeutic agents was reported in WO90/11092 (Oct. 4,1990), in which naked polynucleotides were used to vaccinate vertebrates.
Recently, the coordinate roles of B7 and the major histocompatibility complex (MHC) presentation of epitopes on the surface of antigen presenting cells in activating CTLs for the elimination of tumors was reviewed [Edgington, Biotechnology 11, 1117-1119, 1993]. Once the MHC molecule on the surface of an antigen presenting cell (APC) presents an epitope to a T-cell receptor (TCR), B7 expressed on the surface of the same APC acts as a second signal by binding to CTLA-4 or CD28. The result is rapid division of CD4+helper T-cells which signal CD8+ T-cells to proliferate and kill the APC.
It is not necessary for the success of the method that immunization be intramuscular. Thus, Tang et al., [Nature, 356, 152-154 (1992)] disclosed that introduction of gold microprojectiles coated with DNA encoding bovine growth hormone (BGH) into the skin of mice resulted in production of anti-BGH antibodies in the mice. Furth et al., [Analytical Biochemistry, 205, 365-368, (1992)] showed that a jet injector could be used to transfect skin, muscle, fat, and mammary tissues of living animals. Various methods for introducing nucleic acids was recently reviewed [Friedman, T., Science, 244, 1275-1281 (1989)]. See also Robinson et al., [Abstracts of Papers Presented at the 1992 meeting on Modern Approaches to New Vaccines, Including Prevention of AIDS, Cold Spring Harbor, p92; Vaccine 11, 957 (1993)], where the im, ip, and iv administration of avian influenza DNA into chickens was alleged to have provided protection against lethal challenge. Intravenous injection of a DNA:cationic liposome complex in mice was shown by Zhu et al., [Science 261, 209-211 (Jul. 9, 1993); see also WO93/24640, Dec. 9, 1993] to result in systemic expression of a cloned transgene. Recently, Ulmer et al., [Science 259, 1745-1749, (1993)] reported on the heterologous protection against influenza virus infection by injection of DNA encoding influenza virus proteins.
Wang et al., [P.N.A.S. USA 90, 4156-4160 (May, 1993)] reported on elicitation of immune responses in mice against HIV by intramuscular inoculation with a cloned, genomic (unspliced) HIV gene. However, the level of immune responses achieved was very low, and the system utilized portions of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) promoter and portions of the simian virus 40 (SV40) promoter and terminator. SV40 is known to transform cells, possibly through integration into host cellular DNA. Thus, the system described by Wang et al., is wholly inappropriate for administration to humans, which is one of the objects of the instant invention.
WO 93/17706 describes a method for vaccinating an animal against a virus, wherein carrier particles were coated with a gene construct and the coated particles are accelerated into cells of an animal.
Studies by Wolff et al. (supra) originally demonstrated that intramuscular injection of plasmid DNA encoding a reporter gene results in the expression of that gene in myocytes at and near the site of injection. Recent reports demonstrated the successful immunization of mice against influenza by the injection of plasmids encoding influenza A hemagglutinin (Montgomery, D. L. et al., 1993, Cell Biol., 12, pp.777-783), or nucleoprotein (Montgomery, D. L. et al., supra; Ulmer, J. B. et al., 1993, Science, 259, pp.1745-1749). The first use of DNA immunization for a herpes virus has been reported (Cox et al., 1993, J.Virol., 67, pp.5664-5667). Injection of a plasmid encoding bovine herpesvirus 1 (BHV-1) glycoprotein g IV gave rise to anti-g IV antibodies in mice and calves. Upon intranasal challenge with BHV-1, immunized calves showed reduced symptoms and shed substantially less virus than controls.
Tuberculosis (TB) is a chronic infectious disease of the lung caused by the pathogen Mycobacterium tuberculosis. TB is one of the most clinically significant infections worldwide, with an incidence of 3 million deaths and 10 million new cases each year. It has been estimated that as much as one third of the world""s population may be infected and, in developing countries, 55 million cases of active TB have been reported. Until the turn of the century, TB was the leading cause of death in the United States. But, with improved sanitary conditions and the advent of antimicrobial drugs, the incidence of mortality steadily declined to the point where it was predicted that the disease would be eradicated by the year 2000. However, in most developed countries, the number of cases of active TB has risen each year since the mid-1980""s. Part of this resurgence has been attributed to immigration and the growing number of immunocompromised, HIV-infected individuals. If left unabated, it is predicted that TB will claim more than 30 million human lives in the next ten years. As alarming as these figures may seem, it is of even greater concern that multidrug-resistant (MDR) strains of M. tuberculosis have arisen. These MDR strains are not tractable by traditional drug therapy and have been responsible for several recent outbreaks of TB, particularly in urban centers. Therefore, one of the key components in the management of TB in the long-term will be an effective vaccine [for review see Bloom and Murray, 1993, Science 257, 1055].
M. tuberculosis is an intracellular pathogen that infects macrophages and is able to survive within the harsh environment of the phagolysosome in this type of cell. Most inhaled bacilli are destroyed by activated alveolar macrophages. However, the surviving bacilli can multiply in macrophages and be released upon cell death, which signals the infiltration of lymphocytes, monocytes and macrophages to the site. Lysis of the bacilli-laden macrophages is mediated by delayed-type hypersensitivity (DTH) and results in the development of a solid caseous tubercle surrounding the area of infected cells. Continued DTH causes the tubercle to liquefy, thereby releasing entrapped bacilli. The large dose of extracellular bacilli triggers further DTH, causing damage to the bronchi and dissemination by lymphatic, hematogenous and bronchial routes, and eventually allowing infectious bacilli to be spread by respiration.
Immunity to TB involves several types of effector cells. Activation of macrophages by cytokines, such as interferon-xcex3, is an effective means of minimizing intracellular mycobacterial multiplication. However, complete eradication of the bacilli by this means is often not achieved. Acquisition of protection against TB requires T lymphocytes. Among these, both CD8+ and CD4+ T cells seem to be important [Orme et al, 1993, J. Infect. Dis. 167, 1481]. These cell types secrete interferon-xcex3 in response to mycobacteria, indicative of a Th1 immune response, and possess cytotoxic activity to mycobacteria-pulsed target cells. In recent studies using xcex2-2 microglobulin- and CD8-deficient mice, CTL responses have been shown to be critical in providing protection against M. tuberculosis [Flynn et al, 1992, Proc. Natl. Acad. Sci. USA 89, 12013; Flynn et al, 1993, J. Exp. Med. 178, 2249; Cooper et al, 1993, J. Exp. Med. 178, 2243]. In contrast, B lymphocytes do not seem to be involved, and passive transfer of anti-mycobacterial antibodies does not provide protection. Therefore, effective vaccines against TB must generate cell-mediated immune responses.
Antigenic stimulation of T cells requires presentation by MHC molecules. In order for mycobacterial antigens to gain access to the antigen presentation pathway they must be released from the bacteria. In infected macrophages, this could be accomplished by secretion or bacterial lysis. Mycobacteria possess many potential T-cell antigens and several have now been identified [Andersen 1994, Dan. Med. Bull. 41, 205]. Some of these antigens are secreted by the bacteria. It is generally believed that immunity against TB is mediated by CD8+ and CD4+ T cells directed toward these secreted antigens. In mouse and guinea pig models of TB, protection from bacterial challenge, as measured by reduced weight loss, has been achieved using a mixture of secreted mycobacterial antigens [Pal and Horowitz, 1992 Infect. Immunity 60, 4781; Andersen 1994, Infect. Immunity 62, 2536; Collins, 1994, Veterin. Microbiol. 40, 95].
Several potentially protective T cell antigens have been identified in M. tuberculosis and some of these are being investigated as vaccine targets. Recent work has indicated that the predominant T-cell antigens are those proteins that are secreted by mycobacteria during their residence in macrophages, such as: i) the antigen 85 complex of proteins (85A, 85B, 85C) [Wiker and Harboe, 1992, Microbiol. Rev. 56, 648], ii) a 6 kDa protein termed ESAT-6 [Andersen 1994, Infect. Immunity 62, 2536], iii) a 38 kDa lipoprotein with homology to PhoS [Young and Garbe, 1991, Res. Microbiol. 142, 55; Andersen, 1992, J. Infect. Dis. 166, 874], iv) the 65 kDa GroEL heat-shock protein [Siva and Lowrie, 1994, Imnunol. 82, 244], v) a 55 kDa protein rich in proline and threonine [Romain et al, 1993, Proc. Natl. Acad. Sci. USA 90, 5322], and vi) a 19 kDa lipoprotein [Faith et al, 1991, Immunol. 74, 1].
The genes for each of the three antigen 85 proteins (A, B, and C) have been cloned and sequenced [Borremans et al, 1989, Infect. Immunity 57, 3123; Content et al, Infect. Immunity 59, 3205; DeWit et al 1994, DNA Seq. 4, 267]. In addition, these structurally-related proteins are targets for strong T-cell responses after both infection and vaccination [Huygen et al, 1988, Scand. J. Immunol. 27, 187; Launois et al, 1991, Clin. Exp. Immunol. 86, 286; Huygen et al, 1992, Infect. Immunity 60, 2880; Munk et al, 1994, Infect. Immunity 62, 726; Launois et al, 1994, Infect. Immunity 62, 3679]. Therefore, the antigen 85 proteins are considered to be good vaccine targets.
To test the efficacy of DNA immunization in the prevention of M.tb disease, M.tb protein-coding DNA sequences were cloned into eukaryotic expression vectors. These DNA constructions elicit an immune response when injected into animals. Immunized animals are infected with mycobacteria to evaluate whether or not direct DNA immunization with the gene (or other M.tb genes) could protect them from disease. Nucleic acids, including DNA constructs and RNA transcripts, capable of inducing in vivo expression of M.tb proteins upon direct introduction into animal tissues via injection or otherwise are therefore disclosed. Injection of these nucleic acids may elicit immune responses which result in the production of cytotoxic T lymphocytes (CTLs) specific for M.tb antigens, as well as the generation of M.tb-specific helper T lymphocyte responses, which are protective upon subsequent challenge. These nucleic acids are useful as vaccines for inducing immunity to M.tb, which can prevent infection and/or ameliorate M.tb-related disease.